DESCRIPTION: Tremendous advances have been made in the rational design of inhibitors of many biological processes including protein-protein interactions. However it is often desirable to increase the activity of a protein. Blocking negative regulation pathways is an alternative to directly targeting the protein of interest. The ubiquitin- proteasome mechanism is a key regulator of protein function through rapid and controlled degradation but current proteasome inhibitors have low specificity. With over 600 distinct E3 ubiquitin ligases there is potential for great specificity in targeting protein degradation. As a model to define strategies for selective pharmacological targeting of protein degradation, we will study two Regulator of G protein Signaling proteins, RGS2 and RGS4 that are degraded by different E3 ligase mechanisms. Reduced RGS2 expression is seen in cardiovascular pathologies (hypertension and heart failure) and also plays a role in anxiety and cancer biology. RGS4 has been implicated in heart failure and low levels have been found in brains of schizophrenia patients and in aggressive human breast cancer lines. Thus increasing RGS2 or 4 expressions could have broad pharmacological ramifications. RGS4 is degraded by a single-chain N-end rule E3 ligase mechanism. We recently found that RGS2 degradation utilizes the distinct Cullin-Ring-Ligase E3 mechanism. The complexities and regulation of ubiquitin-proteasome molecular machines make it hard to predict exactly where the druggable soft-spots are likely to be, so broad screening approaches provide a way to detect novel sites of action. By comparing RGS2 and RGS4 regulation, we will explore mechanisms and specificity in E3 targeting. Using a robust, HTS- compatible, cell-based assay to detect RGS2 or RGS4 protein levels we will: 1) identify small molecule enhancers of RGS cellular expression, 2) examine the specificity of their actions in counter- screens, 3) obtain initial structure-activity relationships with commercially available compounds and 4) assess compound mechanisms of action by defining the role of direct compound binding to RGS proteins or to the relevant E3 ligase components. The long term goal of this work is to define strategies for the selective pharmacological targeting of protein degradation. Successful completion of this work will provide mechanistically characterized compound series that enhance RGS2 and RGS4 protein expression and function. Such compounds will serve as chemical probes of protein degradation that would illuminate effects of RGS upregulation in biology and in neuropsychiatric, cardiovascular, and malignant diseases.